The present invention relates to novel non-peptidic compounds having terminal guanidino and carboxyl functional groups, to their preparation and to pharmaceutical compositions comprising them for treatment of several pathological disorders.
The ability of various cell types to adhere to and to interact with other cells or with components of the extracellular matrix (ECM) is essential for maintaining cell functions and tissue integrity via signalling between and within the communicating cells (Springer, 1990; Hynes, 1992; Shimizu et al., 1991). Cellular interactions with soluble or insoluble components of the plasma, the interstitial matrix or the ECM, are carried out primarily via a family of cell-surface receptors designated integrins that are present on most cell types, including lymphocytes, tumor cells and platelets (Ruoslahti, 1991; Hynes, 1992).
The integrins are heterodimeric molecules consisting of an alpha (.alpha.) and a beta (.beta.) subunits which are non-covalently linked. Eleven .alpha. and six .beta. subunits have been identified. The pairing of .alpha. and .beta. subunits exhibits high fidelity in certain tissues and cell types but degenerates in other cases.
The integrins play an important role in integrating the ECM outside the cell with the actin-containing cytoskeleton inside the cell. They are two-headed: the extracellular portion is responsible for the binding of adhesive proteins, in many cases recognizing the ARG-Gly-Asp (RGD; SEQ ID No:1) sequences within these ligands, and the intracellular portion interacts with elements of the cytoskeleton.
The target epitope of several integrin receptors is the RGD sequence, a cell adhesion motif shared by several matrix-associated adhesive glycoproteins, such as fibronectin (FN), vitronectin (VN), fibrinogen, thrombospondin, and von Willebrand factor (Yamada & Kennedy, 1984; Hynes, 1992; Ruoslahti, 1988; D'Souza et al., 1991a).
The best characterized of these proteins is fibronectin, a large and abundant glycoprotein of extracellular matrices and plasma, which serves as a prototype cell adhesion molecule. Fibronectin is a multifunctional protein that supports cell attachment and spreading in eukaryotes and also mediates bacterial cell adhesion. It binds to numerous cell surface and matrix constituents including glycosaminoglycans, heparin, proteoglycans, fibrin and collagen, and triggers a variety of cellular responses (Hynes, 1990).
The tripeptide Arg-Gly-Asp (RGD) was identified as the minimal sequence within the central cell binding domain of fibronectin that mediates cell attachment. The RGD sequence is recognized by several receptors, including the .alpha.IIb.beta.3 (also designated GPIIb-IIIa), .alpha.3.beta.1, .alpha.5.beta.1 (also designated VLA-3 and VLA-5 integrins, respectively) and most of the .alpha.v-containing integrins (Hynes, 1992; Elices et al., 1991; Shimizu et al., 1990). Following cell activation, these receptors mediate RGD-dependent cell-matrix adhesion or cell aggregation (Philips et al., 1991; D'Souza et al., 1991a; Adler et al., 1991). When present in solutions, peptides containing the RGD sequence compete with fibronectin and other RGD-containing matrix proteins for binding to their respective integrin receptors and prevent cell adhesion (Springer, 1990; Ruoslahti and Giancotti, 1989). When immobilized on a surface, short synthetic RGD peptides mimic fibronectin cell-binding properties, but their affinity to their corresponding integrin is about 10.sup.2 -10.sup.3 lower than that of the native ligands (Humphries, et al., 1986).
The RGD motif is not restricted to fibronectin and in fact it is present within more than hundred proteins. In some proteins, cell adhesive activity has been ascribed to the RGD sequence, whereas in most others the RGD sequence appears to be functionally silent. It was found to be a common motif in cell adhesion molecules and it plays a crucial role in platelet aggregation, the immune response, in cancer metastasis, cell migration to tissues, infection of microbial pathogens, gastrulation in Xenopus and Drosophila embryos. Several proteins, which were found to have the sequence RGD expressed on their surface, promote cell attachment in vitro for no apparent physiological reason, also indicating the generality of this binding.
The functional activity of RGD peptides was demonstrated with a variety of cell types. It is particularly significant that RGD peptides are capable of inhibiting the binding of fibrinogen and other related proteins to platelets (small enucleated blood cells), and inhibit platelet aggregation, the cell-cell interaction critical for thrombus formation. This observation indicated that RGD peptides could function as antithrombotic agents.
European Patent Application published under No. EP 410539 describes fibrinogen receptor antagonists which are small cyclic hexapeptides containing the RGD sequence and are claimed to be useful in inhibiting platelet aggregation. European Patent Application published under No. EP 406428 describes synthetic cyclic peptides containing the RGD sequence which are cell adhesion inhibitors useful as platelet aggregation inhibitors and tumor metastasis suppressors. European Patent Application published under No. EP 394326 describes synthetic peptides which incorporate the sequence RGD in a conformationally stabilised form and which may be utilized either for inhibiting binding of adhesion proteins, e.g. vitronectin, or for promoting cell adhesion, e.g. in vivo uses such as coating of medical devices, including prostheses and implants, or in vitro uses in coating of substrates such as cell culture substrates. European Patent Application published under No. EP 384362 describes modified peptides useful as inhibitors of protein-platelet adhesion, cell-cell adhesion and platelet aggregation. International Application published under No. WO 9011297 describes adhesion peptides comprising a biologically active site which is a cell attachment promoting binding site containing the RGD sequence, and a hydrophobic attachment domain, useful for facilitating the attachment of the peptide to solid substrates, e.g., in coating of prosthetic devices to be implanted.
The physiological roles of RGD-mediated recognition may extend beyond these biological processes. Pathogenic microorganisms may adhere to RGD-containing ECM glycoproteins. Thus, Trypanosoma cruzi adheres to fibronectin and peptides modeled from the fibronectin RGD cell attachment domain were shown to inhibit T. cruzi infection (Ouaissi et al., 1986).
Interestingly, several non-ECM related proteins contain the RGD or RGD-like molecules. Among these, the RGD sequence is also found in the transactivation (tat) factor of human immunodeficiency virus type-I (HIV-1). The protein which regulates the viral replication also induces other manifestations of the disease, e.g., Kaposi sarcoma. Soluble tat was shown to bind to several integrins in an RGD-dependent manner (Vogel et al. 1992).
Peptides containing Arg-Ala-Asp (RAD; SEQ ID No:2), Arg-Glu-Asp (RED; SEQ ID No:3), Arg-Phe-Asp (RFD; SEQ ID No:4) and Arg-Tyr-Asp (RYD; SEQ ID No:5) sequences were postulated to interfere with immune functions unrelated to integrins. The Arg-Ala-Asp-Ser (RADS; SEQ ID No:9), Arg-Phe-Asp-Ser (RFDS; SEQ ID No:10) and Arg-Tyr-Asp-Ser (RYDS; SEQ ID No:11) sequences have been postulated to constitute functional adhesiotopes of the CD4 or MHC-I and II molecules, respectively (Mazerolles et al., 1990). Human HLA-DR antigen, present on antigen presenting cells, contains the sequence RYDS and is recognized by the T-cell CD4 antigen. Interference with the CD4-HLA-DR interaction might result in incomplete T cell activation.
Synthetic peptides derived from the human major histocompatibility complex class II antigens (MHC-II) containing the peptide RFDS, and a peptide derived from the immunoglobulin-like amino-terminal domain of the T cell CD4 molecules containing the RADS peptide, were shown to exhibit specific inhibitory effect on antigen-induced HLA class-II-restricted T cell proliferative responses and antibody synthesis (Mazerolles et al, 1988).
The RYDS sequence-has been shown to mimic the RGD cell binding domain of fibrinogen. RYD sequence is comprised as essential part of a CDR-3 (complementarity-determining region) of a monoclonal antibody specific for the binding site of the platelet integrin GPIIb-IIIa, specific for FN, fibrinogen, vitronectin etc. A 12-mer peptide derived from this CDR-3, containing the RYDS site, inhibited RGD-dependent fibrinogen binding to its GPIIb-IIIa receptor (Taub et al., 1989). Streptavidin RYD-sequence has also been shown to mimic the RGD sequence and mediate RGD-dependent cell binding and adhesion of the protein (Alon et al., 1990). Recently, REDV sequence of the alternatively spliced cell-binding domain of FN has been shown to be involved in FN-binding to its non-RGD dependent integrin receptor, .alpha.4.beta.1 (Mould et al., 1991).
Moreover, the inverted peptide Ser-Asp-Gly-Arg (SDGR; SEQ ID No.12) containing the Asp-Gly-Arg (DGR; SEQ ID No:6) sequence was shown to inhibit spreading of BHK cells and chick embryo fibroblasts on vitronectin-coated substrates and on fibronectin-coated substrates. DGR-containing sequences have been suggested to comprise part of the ligand-binding pocket in integrins, implicated in RGD recognition. At any rate, they may interact with RGD-sequences on adhesive proteins and block or inhibit their interactions with integrins (Yamada and Kennedy, 1987).
The use of peptidic RGD analogues presents several drawbacks, mainly the cleavage of the peptidic bond by proteolytic enzymes in vivo. It would therefore be of great advantage to derive functional mimetics resistant to proteolytic digestion to be used as useful tools for interfering with biologic interactions dependent on RGD recognition, such as integrin-mediated cell functions.